LECTURE 3: PROTEINS - "THE WORKHORSES OF A CELL"

Definitions:

Protein

one polypeptide or a group of polypeptides having a distinct folding in three dimensions Polypeptide - fifteen or more amino acids connected by peptide bonds

Amino acids

the basic monomer of polypeptides; all contain an amino group and a carboxylic acid group. The twenty common amino acids are distinguished by the identity of their R (side chain) groups (Figure 2.13). The various types of side chains allow proteins to partake in a variety of chemical reactions.

Peptide bonds link amino acids together in polypeptides (Figure 2.14).

Each protein contains an essentially unique sequence of amino acids. e.g. for a protein that is 300 amino acids long, the probability that its particular sequence would occur at random is (1/20)^300 - not very likely!

To provide chemistries not available in the twenty amino acid side chains, polypeptides may recruit non-protein components - prosthetic groups and coenzymes. Prosthetic groups are tightly bound structures (e.g. heme in hemoglobin protein) Coenzymes are loosely bound and often function as carriers of small molecules (e.g. flavin carries electrons, allowing associated proteins to participate in oxidation-reduction reactions).

I. Protein Structure - there are four levels of structure in proteins:

1. Primary structure - is simply the linear sequence of amino acids of the protein.
   F. Sanger developed methods to determine primary structure, i.e. to sequence proteins.
   L. Pauling predicted that a change (the result of a genetic mutation) in the primary structure of hemoglobin would be responsible for the "molecular disease" sickle cell anemia
2. Secondary structure - refers to the formation of alpha helices and beta sheets, both stabilized by hydrogen bonding between amino groups and carboxyl groups (Figure 2.15) L. Pauling predicted that these structures would occur in proteins before the advent of X-ray crystallography methods for solving the three-dimensional structure of proteins.
3. Tertiary structure - refers to the folding of the protein in three dimensions (Figure 2.16)
4. Quaternary structure - refers to the organization of several associated polypeptides in three dimensions with respect to each other (Figure 2.17). Forces maintaining quaternary structure include hydrogen bonding, salt bridges (ionic interactions), and van der Waals interactions.

II. Denaturation - proteins can be unfolded by disruption of the forces holding them in their native, three-dimensional shape

A. Denaturants include:

1. Certain chemicals - e.g. urea, guanidium hydrochloride - may be reversible
2. Heat denaturation - usually irreversible

B. What is destroyed?

Complete denaturation destroys all levels of protein structure except primary structure. Often, chemical denaturants can be removed and the protein can again assume its native shape - i.e. the protein can renature (Figure 2.18).

C. Key conclusions:

• All the information necessary for protein folding is present in the primary sequence.
• The correct folding of a protein in three dimensions is required for its function.

III. Protein function

A. Catalysis -

Enzymes increase the rate of specific chemical reactions but are not consumed during the reaction (they are regenerated). Their names usually have the suffix "ase".

e.g. Beta-galactosidase - catalyzes the cleavage of the disaccharide lactose, producing two monosaccharides, glucose and galactose:

The equilibrium constant can also be expressed in terms of the forward and reverse rate constants. If the rate of the forward reaction is k1 and the rate of the reverse reaction is k2, then

This makes sense since the amount of product is directly dependent on the forward rate constant and the amount of substrate is directly dependent on the reverse rate constant, so the equilibrium constant (which is really just the ratio between product and substrate at equilibrium) should simply be the ratio between the rate constants governing the formation of product and substrate.

The main point is that enzymes do not alter the equilibrium constant (K) for a reaction, but they do increase the values of the rate constants (k1 and k2), so that reactions that would normally proceed to equilibrium extremely slowly can occur much faster.

Note that the equilibrium constant (K) is not altered. It is 10 whether the enzyme is present or not. However, the rate constants (k1 and k2) are much higher when the enzyme is present.

B. Structural - some proteins provide mechanical support

e.g. silk, collagen in connective tissue

C. Binding

• 1. Non-specific binding - some proteins bind DNA or other proteins in a manner independent of sequence context. E.g. SSB - single-stranded DNA binding protein binds single-stranded DNA regardless of the DNA sequence. E.g. histones - these basic proteins bind the negatively charged backbone of DNA regardless of the DNA sequence.
  
• 2. Specific binding of:
  
• a. Small molecules. E.g. cell surface receptors are used by microorganisms to bind small molecules in the environment that may subsequently elicit a specific response
• b. Nucleic acid sequences. E.g. sigma factor of E. coli RNA polymerase recognizes and binds specific DNA sequences called promoters during the initiation phase of transcription
• c. Other proteins. E.g. binding of an antibody to an antigen (Figure 12.30)
  Antigen - a molecule capable of interacting with specific components of the immune system.
  Antibody - a soluble protein produced by B cells that interacts with antigens (a.k.a. immunoglobulin, IgG)
  
• 3. Antigen-antibody interactions.
  
  Because of the high degree of specificity in an antigen-antibody interaction, antibodies are widely used as probes in the research lab and in clinical labs.